Heart disease is the leading cause of mortality and morbidity in the United States and has been rising dramatically around the world8. Cardiac diseases of the sarcomere, such as HCM and DCM, involve amino acid mutations in one of several myofilament proteins commonly leading to heart failure, and in some cases sudden cardiac death1,2,9. As the number of identified mutants with functional characterization has grown, some patterns have emerged that demonstrate potential similarities in altered contractile properties. For example, most HCM mutations result in increased Ca2+ sensitivity of contractile force in demembranated cardiac muscle1,10-17, while most DCM variants result in decreased Ca2+ sensitivity of force1,18-23. However, the extent of which these alterations in myofilament Ca2+ sensitivity are involved in progression of the diseases is not known. Potential and important interactions between altered myofilament Ca2+ binding and SR function have not been systematically investigated, nor have interactions with other intracellular Ca2+ buffers (e.g., mitochondria) or gene regulation.
Many cardiopathologies, as well as ischemia-reperfusion injury and myocardial infarct result in reduced systolic function due to damage and/or death to a portion of the myocardium that significantly compromises cardiac function. Infarcted hearts often do not meet the cardiovascular demands of the body and attempt to compensate by increasing β-adrenergic activation. Chronic β-adrenergic stimulation, however, exhausts contractile reserves, can elevate diastolic Ca2+ levels, and eventually results in down-regulation of adrenergic responsiveness leading to end-stage heart failure8-10*. Importantly, a number of studies in both animal models and patients have noted alterations in both myofilament11-16 and sarcoplasmic reticulum (SR) and sarcolemmal17-18* protein content and phosphorylation following infarction, which would alter myofilament Ca2+ sensitivity of force and Ca2+ transient release/reuptake. Similar changes have been observed in hearts expressing mutations associated with DCM and HCM35. Although global alterations in hormone levels (such as 3-adrenergic agonists) have often been implicated in these adaptations, the mechanism(s) may be due (at least in part) to intracellular interplay between SR and myofilament proteins.
Cardiac function is compromised in a number of cardiovascular diseases including myocardial infarction, ischemia/reperfusion injury, diabetes, high blood pressure and hypertrophic and dilated cardiomyopathy. These pathophysiological conditions often alter the Ca2+ cycle1, β-adrenergic responsiveness2, and/or the contractile apparatus of cardiomyocytes. To date, therapeutic efforts have focused primarily on increasing [Ca2+]i, which tend to exert a pro-arrhythmogenic effect, impair ventricular filling by slowing diastolic relaxations, and cause SR Ca2+ overload initiating triggered activity19*. Other approaches involving adrenergic agents can have undesirable long-term side-effects, e.g. significant drug actions in non-target areas, pro-arrythmogenic triggered activity, and potential for accelerated progression into heart failure2. Thus, new approaches to combat cardiac dysfunction are desirable.
An alternative approach involves the use of Ca2+ sensitizing compounds that enhance Ca2+ binding to cardiac troponin C (cTnC) and increase contractile strength4. Considerable effort has been made to develop pharmaceutical agents such as calmidazolium, bepridil and levosimenden that increase Ca2+ binding to the N-terminus (site II, the ‘trigger site’) of cTnC and enhance contractile activation. Some drawbacks associated which such compounds include their non-specificity to cTnC and deleterious effects on proteins involved in Ca2+ handling21,22 and other aspects of the excitation-contraction coupling pathway5.
In view of the drawbacks associated with various conventional pharmacological and surgical approaches, which are generally aimed at slowing progression of heart failure as opposed to recovery of function, novel methods of improving cardiac function are needed. The present invention provides more targeted approaches for enhancing cardiac contraction without affecting EC coupling6, i.e. generation of recombinant novel cTnC variants with altered Ca2+ binding properties or in situ production or administration of dATP by ribonucleotide reductase for use as a replacement substrate in cardiac contraction.